A detailed understanding of the processes that control axon growth and guidance is essential for understanding the development of the nervous system and for engineering successful regrowth of severed neurons following spinal cord injury. While many of the molecules responsible for enhancing or inhibiting growth and for attractive and repulsive guidance have been identified, the majority of our knowledge of the mechanisms of axon motility comes from studies on flat, featureless, stiff substrates. There is accumulating evidence, however, that cell motility is sensitive to the structural and mechanical environ- ment. Conversely, there has been significant progress in creating structural features that control axon growth in complex environments in vitro and in vivo, but in most cases the mechanisms responsible for the modulation of motility are not known. The goal of the proposed research is to investigate axonal guidance in rigorously controlled mechanical and structural environments using imaging and analysis tools that will reveal subcellular morphology and dynamics with unprecedented detail. These studies will elucidate the roles of filopodia, focal adhesions, mechanical forces and guidance factors in controlled environments, and provides crucial information for the development of manipulations of the extracellular environment in vivo that impact axon motility in predictable ways, and for the engineering of implant materials to promote nerve regeneration after injury. Our specific aims are to: (1) Determine the mechanisms by which filopodia-collagen fibril interactions promote axon motility and guidance in 3D (2) Assess the effects of matrix composition on growth cone morphology, axon outgrowth, and axon guidance in 3D collagen-I matri- ces, and (3) Test the hypothesis that axons confined to narrow lanes, which mimic fibrillar confinement, are less responsive to non-directed guidance cues. This research will utilize a specialized high-speed, high sensitivity spinning disk confocal microscope system developed in the laboratory of the PI, as well as customized image analysis software. The research is an interdisciplinary collaborative effort involving re- searchers with expertise in cell biology, physics, materials science, and engineering. This effort will produce an entirely new perspective on axon motility and should significantly advance our ability to understand and control axon growth in complex environments. PUBLIC HEALTH RELEVANCE: There is currently no effective treatment for injuries to the central nervous system. The research described in this proposal will provide critical information for guiding interventions to allow nerves to regenerate after spinal injury either through treatment of the injured area or the design of implants that can provide bridges for reconnection.